Different responses of the MIO­M1 Mueller cell line to angiotensin II under hyperglycemic or hypoxic conditions.

Members of the renin-angiotensin aldosterone system (RAAS) are expressed by various retinal tissues including Mueller glial cells. As the RAAS is hypothesized to play an important role in the pathogenesis of diseases that threaten vision, such as diabetic macular edema or retinal vein occlusion, the possible changes induced by exposure of the human cell line MIO-M1, an established model of Mueller cells, to angiotensin II or aldosterone for 6 h under hypoxic and/or hyperglycemic conditions were investigated. The mRNA expression levels of the members of the RAAS were assessed by reverse transcription-quantitative PCR, and the secretion of cytokines was assessed by ELISA. Under hyperglycemic conditions, the mRNA expression levels of the angiotensin-converting enzyme 2 (ACE2), angiotensin II receptors, AT1 and AT2, and the receptor of angiotensin (1-7) MAS1 were significantly higher after exposure to angiotensin II, and the expression of ACE2, AT2, and IL-6 (a marker of inflammation) was significantly increased after treatment with aldosterone; the expression of the other targets investigated remained unchanged. Significantly more IL-6 was secreted by MIO-M1 cells exposed to hyperglycemia and angiotensin. When cells were cultured in a hypoxic environment, additional treatment with aldosterone significantly increased the mRNA expression levels of ACE, but significantly more ACE2 mRNA was expressed in the presence of angiotensin II. Under hypoxic plus hyperglycemic conditions, significantly less ACE but more AT2 was expressed after treatment with angiotensin II, which also led to strongly elevated expression of IL-6. The mRNA expression levels of the angiogenic growth factor VEGF-A and secretion of the encoded protein were notably increased under hypoxic and hypoxic plus hyperglycemic conditions, irrespective of additional treatment with angiotensin II or aldosterone. These findings suggest that angiotensin II induces a pro-inflammatory response in MIO-M1 cells under hyperglycemic conditions despite activation of the counteracting ACE2/MAS1 signaling cascade. However, hypoxia results in an increased expression of angiogenic VEGF-A by these cells, which is not altered by angiotensin II or aldosterone.

Beovu, but not Lucentis impairs the function of the barrier formed by retinal endothelial cells in vitro.

Because rare, but severe adverse effects, i.e. retinal vasculitis or retinal vein occlusion, have been observed after repetitive intravitreal injections of VEGF-A-binding single-chain variable fragment brolucizumab (Beovu), we investigated its possible impact on the barrier formed by immortalized bovine retinal endothelial cells (iBREC) in comparison to that of the VEGF-A-binding Fab fragment ranibizumab (Lucentis). As a measure of stability of the barrier formed by a confluent monolayer of iBREC, we determined the cell index over seven days by continuous electric cell-substrate impedance measurements: Beovu but not Lucentis indeed significantly lowered the cell index, evident about 1.5 days after its addition, pointing to barrier impairment. Early after addition of Beovu, amounts of the integrins α5 and ß1-subunits of the fibronectin receptor-had changed in opposite ways, suggesting an effect on cell adhesion due to hindered dimer formation. After exposure for eight days to Beovu, levels of claudin-1-an essential part of the iBREC barrier-were significantly lower, less claudin-1 was located at the plasma membrane after exposure to the VEGF-A antagonist for five days. Beovu did not induce secretion of inflammatory cytokines or VEGF-A. Interestingly, polysorbate-80-component of Beovu-but not polysorbate-20-in Lucentis-slightly, but significantly lowered the cell index, also associated with reduced claudin-1 expression. In summary, our results indicate that Beovu changes the behavior of retinal endothelial cells, thus providing an alternative "non-immunological" explanation for the most relevant of observed side effects.

Impairment of the Retinal Endothelial Cell Barrier Induced by Long-Term Treatment with VEGF-A165 No Longer Depends on the Growth Factor's Presence.

As responses of immortalized endothelial cells of the bovine retina (iBREC) to VEGF-A165 depend on exposure time to the growth factor, we investigated changes evident after long-term treatment for nine days. The cell index of iBREC cultivated on gold electrodes-determined as a measure of permeability-was persistently reduced by exposure to the growth factor. Late after addition of VEGF-A165 protein levels of claudin-1 and CD49e were significantly lower, those of CD29 significantly higher, and the plasmalemma vesicle associated protein was no longer detected. Nuclear levels of ß-catenin were only elevated on day two. Extracellular levels of VEGF-A-measured by ELISA-were very low. Similar to the binding of the growth factor by brolucizumab, inhibition of VEGFR2 by tyrosine kinase inhibitors tivozanib or nintedanib led to complete, although transient, recovery of the low cell index when added early, though was inefficient when added three or six days later. Additional inhibition of other receptor tyrosine kinases by nintedanib was similarly unsuccessful, but additional blocking of c-kit by tivozanib led to sustained recovery of the low cell index, an effect observed only when the inhibitor was added early. From these data, we conclude that several days after the addition of VEGF-A165 to iBREC, barrier dysfunction is mainly sustained by increased paracellular flow and impaired adhesion. Even more important, these changes are most likely no longer VEGF-A-controlled.

Blocking of VEGF-A is not sufficient to completely revert its long-term effects on the barrier formed by retinal endothelial cells.

The VEGF-A-induced functional impairment of the barrier formed by retinal endothelial cells (REC) can be prevented and even - at least temporarily - reverted by trapping the growth factor in a complex with a VEGF-binding protein or by inhibiting the activity of the VEGF receptor 2 (VEGFR2). In an approach to emulate the clinically relevant situation of constant exposure to effectors, we investigated (1) whether prolonged exposure to VEGF-A165 for up to six days results in a different type of disturbance of the barrier formed by immortalized bovine REC (iBREC) and (2) whether alterations of the barrier induced by VEGF-A165 can indeed be sustainably reverted by subsequent treatment with the VEGF-A-binding proteins ranibizumab or brolucizumab. As a measure of barrier integrity, the cell index (CI) of iBREC cultivated on gold electrodes was monitored continuously. CI values declined shortly after addition of the growth factor and then remained low for more than six days over which considerable amounts of both extra- and intracellular VEGF-A were measured. Interestingly, the specific VEGFR2 inhibitor nintedanib normalized the lowered CI when added to iBREC pre-treated with VEGF-A165 for one day, but failed to do so when cells had been exposed to the growth factor for six days. Expression of the tight junction (TJ) protein claudin-5 was unchanged early after addition of VEGF-A165 but higher after prolonged treatment, whereas decreased amounts of the TJ-protein claudin-1 remained low, and increased expression of the plasmalemma vesicle-associated protein (PLVAP) remained high during further exposure. After two days, the characteristic even plasma membrane stainings of claudin-1 or claudin-5 appeared weaker or disordered, respectively. After six days the subcellular localization of claudin-5 was similar to that of control cells again, but claudin-1 remained relocated from the plasma membrane. To counteract these effects of VEGF-A165, brolucizumab or ranibizumab was added after one day, resulting in recovery of the then lowered CI to normal values within a few hours. However, despite the VEGF antagonist being present, the CI declined again two days later to values that were just slightly higher than without VEGF inhibition during further assessment for several days. At this stage, neither the supernatants nor whole cell extracts from iBREC treated with VEGF-A165 and its antagonists contained significant amounts of free VEGF-A. Treatment of VEGF-A165-challenged iBREC with ranibizumab or brolucizumab normalized expression of claudin-1 and claudin-5, but not completely that of PLVAP. Interestingly, the characteristic VEGF-A165-induced relocalization of claudin-1 from the plasma membrane was reverted within one day by any of the VEGF antagonists, but reappeared despite their presence after further exposure for several days. Taken together, barrier dysfunction induced by VEGF-A165 results from deregulated para- and transcellular flow but the precise nature or magnitude of underlying changes on a molecular level clearly depend on the time of exposure, evolving into a stage of VEGF-A165-independent barrier impairment. These findings also provide a plausible explanation for resistance to treatment with VEGF-A antagonists frequently observed in clinical practice.

Type of culture medium determines properties of cultivated retinal endothelial cells: induction of substantial phenotypic conversion by standard DMEM.

Contradictory behavior of microvascular retinal endothelial cells (REC) - a reliable in vitro model to study retinal diseases - have recently been reported which might result from cultivating the cells in standard DMEM not optimized for this cell type. Therefore, we studied DMEM's effects on phenotype and behavior of immortalized bovine REC. Cells were cultivated in endothelial cell growth medium (ECGM) until a confluent monolayer was reached and then further kept for 1-4 days in ECGM, DMEM, or mixes thereof all supplemented with 5% fetal bovine serum, endothelial cell growth supplement, 90 µg/ml heparin, and 100 nM hydrocortisone. Within hours of cultivation in DMEM, the cell index - measured to assess the cell layer's barrier function - dropped to ~5% of the initial value and only slowly recovered, not only accompanied by stronger expression of HSP70 mRNA and secretion of interleukin-6, but also by lower expressions of tight junction proteins claudin-5, claudin-1 or of the marker of cell type conversion caveolin-1. Altered subcellular localizations of EC-typic claudin-5, vascular endothelial cadherin and von Willebrand factor were also observed. Taken together, all experiments with (retinal) EC cultivated in common DMEM need to be interpreted very cautiously and should at least include phenotypic validation.

Transport and fate of aflibercept in VEGF-A165-challenged retinal endothelial cells.

Retinal vessels are at least in part involved in clearing of Fc terminus-containing proteins from the vitreous. In vitro, the Fc fusion protein aflibercept is transported through a monolayer of unchallenged immortalized bovine retinal endothelial cells (iBREC), mediated by the neonatal Fc receptor (FcRn), but part of the Fc fusion protein is also degraded. Aflibercept's target VEGF-A not only enhances the permeability of REC by destabilization of tight junctions (TJs) thereby allowing for paracellular flow, it may also lower the intracellular stability of the Fc fusion protein by changing its binding properties to the FcRn. Therefore, we investigated the transport and fate of aflibercept in VEGF-A165-challenged iBREC. All cell culture media were supplemented with 5% fetal bovine serum (FBS) as its absence results in accumulation of aflibercept in iBREC due to deregulated expression of transport proteins. Early after exposure of a confluent iBREC monolayer cultivated on gold electrodes to 5% FBS, the cell index (CI) - assessed as a measure of barrier function, cell viability and cell adhesion - transiently declined but recovered again within a few hours to high values. These values remained stable for several days associated with a strong expression of the TJ-protein claudin-1, indicative of a functional barrier formed by the iBREC monolayer. Transient changes of the plasma membrane localizations of claudin-5 and vascular endothelial cadherin - both important for regulation of paracellular flow - accompanied the transient reduction of the CI not prevented by VEGF-binding proteins. Treatment of iBREC with 50 ng/ml VEGF-A165 for one day resulted in a strong and persistent decline of the CI associated with a low expression level of the TJ-protein claudin-1; reversion to normal values was complete one day after aflibercept's addition at a final concentration of 250 µg/ml. Expressions of other proteins involved in regulation of paracellular flow or transcellular transport were not significantly changed. More aflibercept passed through the monolayer of iBREC cultivated on permeable membrane inserts pretreated with VEGF-A for one day, but this was not affected by a FcRn-inhibiting antibody. Subcellular localization of aflibercept was hardly changed in VEGF-A-exposed iBREC 3 h after its addition to the cells; inhibition of (non)-lysosomal or proteasomal proteases then only weakly affected the amount of internalized aflibercept. iBREC also internalized VEGF-A which was barely detectable as early as 2 h after addition of aflibercept. In contrast, blocking the tyrosine kinase activity of VEGF receptor(s) did not prevent VEGF-A's uptake. Inhibition of cellular proteases strongly increased the amount of internalized VEGF-A in the absence and presence of the Fc fusion protein. We therefore conclude that a FcRn-mediated transport plays a minor role in aflibercept's passage through a leaky barrier of REC. Even early after addition of aflibercept to VEGF-A-exposed iBREC, the levels of free intracellular VEGF-A are low, as aflibercept likely prevents binding of VEGF-A to its receptor. Interestingly, the growth factor's detrimental effects still persist for nearly one day.

Sitagliptin and the Blood-Retina Barrier: Effects on Retinal Endothelial Cells Manifested Only after Prolonged Exposure.

Inhibitors of dipeptidyl peptidase-4 (DPP-4) are widely used to treat diabetes mellitus, but data concerning their effects on the barrier stability of retinal endothelial cells (REC) in vivo and in vitro are inconsistent. Therefore, we studied whether the barrier properties of immortalized endothelial cells of the bovine retina (iBREC) were affected by the inhibitors of DPP-4 sitagliptin (10-1000 nM) and diprotin A (1-25 µM). Their effects were also investigated in the presence of VEGF-A165 because diabetic patients often develop macular edema caused by VEGF-A-induced permeability of REC. To detect even transient or subtle changes of paracellular and transcellular flow as well as adhesion of the cells to the extracellular matrix, we continuously monitored the cell index (CI) of confluent iBREC grown on gold electrodes. Initially, the CI remained stable but started to decline significantly and persistently at 40 h or 55 h after addition of sitagliptin or diprotin A, respectively. Both inhibitors did not modulate, prevent, or revert the persistent VEGF-A165-induced reduction of the CI. Interestingly, sitagliptin and diprotin A increased the expression of the tight-junction protein claudin-1 which is an important component of a functional barrier formed by iBREC. In contrast, expressions of CD29-a subunit of the fibronectin receptor-or of the tetraspanin CD9 were lower after extended treatment with the DPP-4 inhibitors; less of the CD9 was seen at the plasma membrane after prolonged exposure to sitagliptin. Because both associated proteins are important for adhesion of iBREC to the extracellular matrix, the observed low CI might be caused by weakened attachment of the cells. From our results, we conclude that extended inhibition of DPP-4 destabilizes the barrier formed by microvascular REC and that DPP-4 inhibitors like sitagliptin do not counteract or enhance a VEGF-A165-induced barrier dysfunction as frequently observed in DME.

VEGF receptor 2 inhibitor nintedanib completely reverts VEGF-A165-induced disturbances of barriers formed by retinal endothelial cells or long-term cultivated ARPE-19 cells.

Various severe ocular diseases are associated with an elevated intravitreal expression of VEGF-A which increases the permeability of retinal endothelial cells (REC) or retinal pigment epithelial (RPE) cells in vivo and in vitro. Inhibition of VEGF receptor 2 (VEGFR2) is sufficient to completely prevent VEGF-A165-induced dysfunctions of barriers formed by long-term cultivated, immortal human ARPE-19 cells or immortalized bovine retinal endothelial cells (iBREC). Extended exposure to VEGF-A could result in additional activation of other growth factor receptors, potentially promoting synergistic effects of corresponding factors on various cellular processes including angiogenesis. Based on these observations, we investigated whether blocking of VEGFR2 is also sufficient to revert VEGF-A-induced changes of the barriers consisting of iBREC (i.e. inner blood-retina barrier) or ARPE-19 cells (i.e. outer blood-retina barrier) in vitro. Alterations of confluent monolayers' properties induced by treatment with VEGF-A165 for one day followed by addition of small molecule inhibitors of the VEGFR2 were determined by continuous cell index (CI) measurements using the microelectronic biosensor system for cell-based assays xCELLigence. VEGF-A165 induced a long-lasting drop of the otherwise high CI of iBREC accompanied by reduced expression of the tight junction (TJ) protein claudin-1 and subtle changes of the plasma membrane localizations of TJ-protein claudin-5 and of vascular endothelial cadherin. Blocking mainly VEGFR2 with 10 nM nintedanib, 10 nM tivozanib or 500 nM ZM323881 efficiently reverted these changes within one day; higher concentrations of nintedanib or additional inhibition of neuropilin-1 were not superior. Interestingly, the CI of short-term cultivated, confluent ARPE-19 cells slightly increased in the presence of VEGF-A165, but was not changed by nintedanib. In contrast, VEGF-A165 markedly reduced the transepithelial electrical resistance of ARPE-19 cells cultivated on porous membrane inserts for three weeks, which was also accompanied by a significant loss of the then strongly plasma membrane-expressed TJ-protein ZO-1. These alterations were completely reverted within one day by 10 nM nintedanib of which higher concentrations were not superior. None of the inhibitors tested diminished the strong barrier properties of iBREC or long-term cultivated ARPE-19 cells. Taken together, inhibition of VEGFR2 efficiently reverts VEGF-A165-induced barrier disturbances of both cell types forming and regulating the inner and outer blood-retina barrier. As synergistic actions of growth factors seem to play only a minor role in inducing a barrier dysfunction, specific inhibition of VEGFR2 could be an interesting option to treat VEGF-A-induced macular edema without obvious effects on vitality and functions of REC and RPE cells.

Fate of the Fc fusion protein aflibercept in retinal endothelial cells: competition of recycling and degradation.

PURPOSE: Intravitreal injection of the VEGF-binding protein aflibercept is widely used to treat various ocular diseases. In vitro, immortalized bovine retinal endothelial cells (iBREC) take up and transport aflibercept through the cell layer in a serum-dependent manner, likely mediated through the neonatal Fc receptor (FcRn), but degradation of the Fc domain-containing protein might be a competing intracellular process. Therefore, aflibercept's associations with proteins either involved in FcRn-mediated transport or in the lysosomal pathway were studied. METHODS: Confluent iBREC pre-cultivated with or without FBS were exposed for 4 h to in vivo achievable 250 µg/ml aflibercept, before cells were harvested for immunofluorescence staining or preparation of protein extracts. Intracellular localization of aflibercept and putative co-localizations with proteins involved in transport of IgG/FcRn complexes, i.e., endosomal Rab4 and Rab11, components of the cytoskeleton, motor proteins, or with marker proteins characteristic of multivesicular bodies or lysosomes were assessed by co-immunofluorescence stainings. Amounts of expressed endogenous proteins and of internalized aflibercept were determined by Western blot analyses. RESULTS: Aflibercept-specific perinuclear staining overlapped with that of the motor protein dynein whereas double staining with an anti-kinesin antibody resulted in a patchy pattern. In addition, aflibercept was typically present close to microtubules and often co-localized with α-tubulin. Rab4 and Rab11 stainings partly overlapped with the perinuclear staining of aflibercept whereas co-localization with Rab7 (in late endosomes/lysosomes) was only rarely seen. Interestingly, aflibercept but not the IgG bevacizumab broadly co-localized with the cation-independent mannose 6-phosphate receptor characteristic of multivesicular endosomes. In accordance with partial degradation beside transcytosis, the amount of intracellular aflibercept increased when cells were treated with protease inhibitors MG-132 or MG-101. Serum-deprived iBREC expressed less Rab11 and dynein but slightly more Rab4. CONCLUSION: After uptake by iBREC, aflibercept is present in organelles associated with FcRn-mediated transport, but part of the protein is subject to degradation. Transport inhibition of aflibercept during cultivation without FBS is likely a consequence of an attenuated exocytosis due to decreased expression of Rab11.

Inhibition of Single Routes of Intracellular Signaling is Not Sufficient to Neutralize the Biphasic Disturbance of a Retinal Endothelial Cell Barrier Induced by VEGF-A165.

BACKGROUND/AIMS: Hallmark of diabetic macular edema is the enhanced permeability of retinal endothelial cells (REC) induced by vascular endothelial growth factor (VEGF-A165), which acts through activating specific receptors. To improve the predictability of inhibitors' potentials to block harmful effects of VEGF-A165, we investigated if its signaling pathways triggered in REC are redundant. METHODS: Immortalized bovine REC monolayers were treated with inhibitors specific for various protein kinases in combination with VEGF-A165. Permeability was monitored continuously by measurements of the cell index (CI) to reveal even subtle and transient changes. Expression of tight junction (TJ) proteins was determined as additional indicator of barrier stability. RESULTS: After a sharp but transient CI drop caused by VEGF-A165 early after its addition, further exposure resulted in a continuous CI decline over several days associated with loss of TJ protein claudin-1. Both phases were blocked by inhibition of VEGF receptor 2. Tested inhibitors of intracellular kinases had a limited or no effect, or were efficient only in certain phases of exposure to VEGF-A165, e.g. inhibiting protein kinase C only prevented the early response. High concentrations of some inhibitors even resulted in VEGF-independent barrier destabilization. CONCLUSIONS: Specific kinase inhibitors differently affect VEGF-A165-triggered processes in distinct phases of its action. VEGF-A165-initiated signaling is redundant and blocking of key proteins of single pathways is not sufficient to suppress REC barrier breakdown.

Neonatal Fc receptor FcRn is involved in intracellular transport of the Fc fusion protein aflibercept and its transition through retinal endothelial cells.

Retinal endothelial cells (REC) likely contribute to the clearance of intravitreally injected IgG. Because this is of high relevance to the pharmacokinetic assessment of the widely used therapeutic Fc fusion protein aflibercept, we studied its transport through immortalized bovine REC (iBREC) in detail. For shuttling of IgG or Fc fusion proteins like aflibercept, endothelial cells use the highly conserved neonatal Fc receptor (FcRn) also expressed in iBREC where it is down regulated by serum depletion. Therefore, we focused on studying intracellular localization and transport of aflibercept under conditions affecting its interaction with the FcRn. Intracellular localization of aflibercept was assessed by Western-blot analyses of subcellular protein fractions or by immunofluorescence staining. After uptake in a temperature-dependent process, aflibercept co-localized with early endosomes, which harbor FcRn. Similar amounts of aflibercept were co-extracted with proteins from membranes/organelles irrespectively of the amount of FBS in the culture medium. Lowering the concentration of FBS resulted in a strong, but reversible association with cytoskeletal proteins suggesting a block in intracellular transport. In accordance with this finding, aflibercept's transport through an iBREC monolayer grown on porous membrane inserts was markedly delayed in the absence of FBS in the culture medium indicating that aflibercept is taken up but not exocytosed under these conditions. Transcytosis of aflibercept was also strongly delayed by inhibition of phosphatidylinositol 3-kinase with LY294002, which affects FcRn-mediated IgG transport. A similar inhibition of aflibercept's transport was observed with IgG-binding proteins (i.e. protein A or protein G) that block interaction between FcRn and aflibercept. Interfering with aflibercept's binding to the FcRn with protein A (or protein G) or the inhibitory FcRn-specific monoclonal antibody 1G3 resulted in a reduced amount of intracellular aflibercept. Taken together, our results strongly suggest that FcRn is involved in transport of aflibercept through REC in vitro.

Pre-Analytical Parameters Affecting Vascular Endothelial Growth Factor Measurement in Plasma: Identifying Confounders.

BACKGROUND: Vascular endothelial growth factor-A (VEGF-A) is intensively investigated in various medical fields. However, comparing VEGF-A measurements is difficult because sample acquisition and pre-analytic procedures differ between studies. We therefore investigated which variables act as confounders of VEGF-A measurements. METHODS: Following a standardized protocol, blood was taken at three clinical sites from six healthy participants (one male and one female participant at each center) twice one week apart. The following pre-analytical parameters were varied in order to analyze their impact on VEGF-A measurements: analyzing center, anticoagulant (EDTA vs. PECT / CTAD), cannula (butterfly vs. neonatal), type of centrifuge (swing-out vs. fixed-angle), time before and after centrifugation, filling level (completely filled vs. half-filled tubes) and analyzing method (ELISA vs. multiplex bead array). Additionally, intrapersonal variations over time and sex differences were explored. Statistical analysis was performed using a linear regression model. RESULTS: The following parameters were identified as statistically significant independent confounders of VEGF-A measurements: analyzing center, anticoagulant, centrifuge, analyzing method and sex of the proband. The following parameters were no significant confounders in our data set: intrapersonal variation over one week, cannula, time before and after centrifugation and filling level of collection tubes. CONCLUSION: VEGF-A measurement results can be affected significantly by the identified pre-analytical parameters. We recommend the use of CTAD anticoagulant, a standardized type of centrifuge and one central laboratory using the same analyzing method for all samples.

Safety of monoclonal antibodies and related therapeutic proteins for the treatment of neovascular macular degeneration: addressing outstanding issues.

INTRODUCTION: The vascular endothelial growth factor (VEGF) inhibitors most widely used to treat neovascular age-dependent macular degeneration (nAMD) are different proteins with structural features potentially relevant to adverse effects (AEs). Two of these are also established in cancer therapy (with higher dosages and AEs). The importance of ocular AE and extraocular activities is still a subject of controversy and ongoing research. AREAS COVERED: Potential risks of intraocular VEGF inhibition based on prospective studies, in vitro investigations, pharmacokinetics, and hints from anti-cancer treatment. EXPERT OPINION: nAMD is a frequently observed chronic clinical condition severely affecting the visual function of elderly persons. Intravitreal injection of VEGF-inactivating proteins is highly effective to prevent loss of vision. Anti-VEGF therapy is well tolerated, and low rates of ocular and systemic AEs in smaller trials suggest a very high benefit/risk ratio. The proteins established in nAMD therapy show similar efficacies. In the controversy over the off-label use of bevacizumab purely on grounds of much lower cost, the small, but potentially relevant differences between the available drugs are easily either dramatized (by pharmaceutical companies) or trivialized (by health insurances) and even political interference is involved. Facing the lack of a convincing body of evidence regarding safety, further long-term study results seem necessary.

The Protein Kinase C Inhibitor: Ruboxistaurin.

The isozyme protein kinase C (PKC) ß is involved in several processes that are deregulated in different retinal cell types by hyperglycemia. This family of serine/threonine-specific protein kinases comprises several different members, which differ in their structure, cofactor requirement and substrate specificity. Therefore, PKCß was considered a valuable target for therapeutic intervention. However, there is now evidence that even inhibition of different PKC isozymes is not sufficient to normalize vascular endothelial growth factor (VEGF)-induced barrier damage of retinal endothelial cells. On the other hand, PKCß inhibition prevents hyperglycemia-induced VEGF expression in retinal pericytes, suggesting that PKC inhibitors should be administered before increased VEGF expression is established in the diabetic retina. Although initial studies have indicated that the treatment of diabetic patients with ruboxistaurin, a specific inhibitor of PKCß, may reduce visual loss in patients with diabetic retinopathy, the overall benefit seems to be small.

Internalization of bevacizumab by retinal endothelial cells and its intracellular fate: Evidence for an involvement of the neonatal Fc receptor.

Bevacizumab is one of the VEGF-binding proteins that are established in clinical practice to treat various ocular diseases. In view of therapeutic long-term application, potential accumulation of the antibody in retinal cells gave reason for safety concerns. Internalization of considerable amounts of bevacizumab by retinal endothelial (REC) and pigment epithelial cells has been observed which may affect their important functions. Therefore we investigated the transport and intracellular localization of bevacizumab in immortalized bovine REC (iBREC) in detail, considering possible roles of vesicles and receptors mediating uptake and intracellular transport. By performing transcytosis assays with iBREC monolayers cultivated on porous membrane inserts, we demonstrated that bevacizumab was transported efficiently through a tight monolayer from the lower to the upper chamber or vice versa. When added to the lower chamber in excess, the internalized antibody was transported through the cells, but it was also recycled to be set free at the same side of the cell into a bevacizumab-free environment. The rates of both processes strongly depended on the concentration of fetal bovine serum (FBS) in the environment. This observation is important because in vivo REC might be exposed to varying amounts of serum, e.g. in patients with macular edema. FBS also affected the intracellular localization of bevacizumab as shown by analyses of subcellular fractions and direct immunofluorescence staining. When iBREC were cultivated in low-serum medium, most of the antibody was found in the fraction of cytoskeleton proteins and spots of high intensity of bevacizumab-specific staining close to the nuclei were observed. Cultivation in medium with FBS resulted in internalized bevacizumab predominately found in the membrane/organelle fraction in addition to its weaker association with proteins from the cytoskeleton and uniform staining of the cell. Bevacizumab-specific staining close to the cytoskeleton proteins α-tubulin or vimentin was also observed. Accumulation and association of the antibody with the cytoskeleton induced by serum reduction could be reversed by subsequent FBS addition. In uptake and transport of bevacizumab vesicles and binding to a receptor seems to be involved: Internalization was strongly temperature-dependent which ruled out paracellular passage and a fraction of the internalized bevacizumab was associated with early endosomes. Protein A inhibited transcytosis and affected intracellular localization suggesting a key role of the neonatal Fc receptor (FcRn). Interestingly, FcRn expression was decreased when iBREC were cultivated without FBS. Our results suggest this pathway of bevacizumab uptake and transition through iBREC: Independent of serum, bevacizumab is taken up through a nonspecific mechanism. The subsequent sorting into transport vesicles depends on the presence of serum as regulator of FcRn expression. Without sufficient amounts of the receptor being expressed, a likely obstructed exocytosis results in intracellular accumulation and an increased association with cytoskeleton proteins. Interaction of substantial amounts of bevacizumab with the cytoskeleton may be the reason for under these conditions suppressed migration of iBREC. If long-term therapies by intravitreal injection lead to accumulation of bevacizumab in REC in vivo and potentially harmful consequences, will have to be revealed by future investigations.

Binding of VEGF-A is sufficient to abrogate the disturbing effects of VEGF-B together with VEGF-A on retinal endothelial cells.

PURPOSE: Inhibition of vascular endothelial growth factor (VEGF) is a promising strategy to treat retinal complications of diabetes. In contrast to VEGF-A binding ranibizumab, aflibercept also binds to other members of the VEGF family including VEGF-B, but potential effects of this factor on permeability and angiogenic processes are unclear. Therefore, we studied how VEGF-B variants as single agents or together with VEGF-A165 might affect proliferation, migration, or barrier function of retinal endothelial cells (REC). Also investigated was the normalization of REC properties with both VEGF-inhibitors to explore if additional targeting of VEGF-B is relevant. METHODS: Stimulation of proliferation or migration of immortalized bovine REC (iBREC) and disturbance of their barrier by exposure to VEGF-B variants (as single factors or together with VEGF-A165) was determined with or without VEGF-binding proteins being added. Permeability of iBREC was assessed by measuring their transendothelial resistance (TER) and expression of the tight junction protein claudin-1. RESULTS: VEGF-B167 and VEGF-B186 enhanced proliferation of iBREC but these isoforms did not affect cell migration. Interestingly, ranibizumab completely blocked both migration and proliferation induced by VEGF-A plus VEGF-B. Both VEGF-B variants did also not affect barrier function or claudin-1 expression in a normal or high-glucose environment. Accordingly, binding VEGF-A was enough to normalize a reduced TER and reinstate claudin-1 lost during treatment with this factor in combination with VEGF-B. CONCLUSIONS: Important properties and functions of REC seem not to be affected by any VEGF-B variant and targeting the key factor VEGF-A is sufficient to normalize growth factor-disturbed cells of this type.

Capacity of aflibercept to counteract VEGF-stimulated abnormal behavior of retinal microvascular endothelial cells.

Members of the vascular endothelial growth factor (VEGF) family differently regulate processes in retinal endothelial cells (REC) which are crucially involved in the pathogenesis of diabetic retinopathy: Both, VEGF-A and placenta growth factor (PlGF), stimulate proliferation of primary and immortalized bovine REC ((i)BREC) but only VEGF-A165 stimulates their migration. Diabetic macular edema is most likely a consequence of an elevated permeability of REC which can be induced by VEGF-A, but not by PlGF. Binding of VEGF-A by the antibody fragment ranibizumab is sufficient to completely restore or prevent VEGF-A-induced disturbance of the iBREC barrier or migration of these cells without affecting the basal processes. This was observed even in the presence of other growth factors when surplus proliferation was only partly blocked. The recombinant protein aflibercept (VEGF-trap) not only binds very strongly to VEGF-A, but - in contrast to ranibizumab - also recognizes PlGF. In this study, we investigated whether this additional targeting of PlGF also results in better inhibition of growth factor-induced proliferation and migration, and disturbance of the iBREC barrier. In addition, uptake of aflibercept by iBREC and potential functional consequences were examined. In accordance with its binding specificity, aflibercept strongly and specifically inhibited iBREC proliferation stimulated with VEGF-A, PlGF or a combination of these factors. By treatment with aflibercept at therapeutically achievable concentrations, VEGF-A-stimulated iBREC migration was reduced not only to normal values but driven below the basal level. However, the VEGF-A binding humanized antibody bevacizumab as well as the unrelated control antibody rituximab also inhibited basal or VEGF-A stimulated migration at clinically relevant concentrations, suggesting an effect of high amounts of IgG domain-containing proteins which does not depend on their binding specificity. However, aflibercept specifically blocked VEGF-A stimulated migration at lower concentration without influencing basal processes. Effects on permeability were determined by measuring transendothelial resistance (TER) of iBREC (±VEGF-A165) and their expression of the tight junction protein claudin-1. The VEGF-A-disturbed barrier was completely restored by treatment with ≤25 µg/ml aflibercept of which even much higher concentrations did not interfere with normal barrier function. Uptake of aflibercept by iBREC - analyzed by Western blot - was observed after 1 h of treatment and the amount further increased during prolonged incubation. Most of the internalized aflibercept was present in subcellular fractions of proteins assigned to the membranes and organelles, but it was also detected in the fraction consisting of cytoskeletal proteins. Co-immunofluorescence staining showed aflibercept absorbed by iBREC to be localized in or close to the Golgi apparatus. Aflibercept at high concentrations interferes with an important normal iBREC function, but prevents and restores VEGF-A-induced disturbances at considerably lower concentrations. Therefore, reduction of the doses administered in DR and DME therapy might be considered.

VEGF but not PlGF disturbs the barrier of retinal endothelial cells.

Elevated permeability of retinal endothelial cells (REC), as observed in diabetic retinopathy (DR), is induced by extended exposure to ≥25 ng/ml vascular endothelial growth factor A165 (VEGF165) for up to 3 d and this effect is more pronounced when equimolar amounts of basic fibroblast growth factor (bFGF) and insulin-like growth factor (IGF-1) are present. Down-regulation of the tight-junction protein claudin-1 and its loss from the plasma membrane is associated with induced higher permeability, whereas other tight-junction proteins (e.g. claudin-3, claudin-5, ZO-1) show only subtle changes in our experimental setting. Using immortalized bovine REC (iBREC) as a well-established model, we investigated effects of other members of the VEGF family, i.e. VEGF121, placental growth factor (PlGF-1 and PlGF-2) and viral VEGF-E which activate different sets of VEGF receptors, on barrier function after extended treatment: iBREC were incubated with 1-100 ng/ml of the growth factors for up to 2 days before barrier function was assessed by measuring transendothelial resistance (TER). Presence of TJ-proteins was determined by western blot analyses and immunofluorescence staining. Similar experiments were performed to evaluate whether the primary actions of PlGF-1, PlGF-2 or VEGF121 are modulated by bFGF or IGF-1 when all growth factors (each at 25 ng/ml, but 10 ng/ml IGF-1) act simultaneously at equimolar concentrations. We also studied the potential normalization of the barrier disturbed with combinations of growth factors by addition of the VEGF-specific Fab fragment ranibizumab or the recombinant protein aflibercept which binds VEGF and PlGF. Whereas 1 ng/ml VEGF-E were sufficient to impair the iBREC barrier, a higher concentration of 100 ng/ml VEGF121 was needed to reduce TER and expression of claudin-1 over 2 days. By PlGF-1 or PlGF-2, the barrier was not affected even at the highest concentration tested (100 ng/ml) and these factors also did not modulate the effect of VEGF165. The weak barrier derangement caused by VEGF121 was slightly enhanced by bFGF and IGF-1. After induction of the barrier breakdown with various combinations of all growth factors included in the study, normal TER and claudin-1 expression was re-established by ranibizumab. Both VEGF inhibitors ranibizumab and aflibercept similarly reinstated lost claudin-1, even when applied at a small fraction of the clinically relevant concentrations. These results show that VEGF-A, but not PlGF impairs the barrier function of iBREC and that the longer isoform VEGF165 is more potent than VEGF121. To induce barrier dysfunction in iBREC, activation of VEGF receptor 2 - probably in concert with neuropilin-1 - seems to be sufficient because VEGF-E and VEGF165, but not PlGF-1/-2 reduced TER or claudin-1 expression.

Ranibizumab efficiently blocks migration but not proliferation induced by growth factor combinations including VEGF in retinal endothelial cells.

BACKGROUND: Proliferation and migration of retinal endothelial cells (REC) are associated with the development of proliferative diabetic retinopathy. REC proliferation is stimulated by isoforms of vascular endothelial growth factor-A (i.e., VEGF121 and VEGF165), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1) of which VEGF165 also enhances migration of REC. Effects induced by VEGF-A can be blocked with ranibizumab, a VEGF-binding Fab fragment used in therapy of diabetic macular edema. In this study, we investigated potential angiogenic effects of placental growth factors (PlGF-1, PlGF-2) as other members of the VEGF family and whether the primary action of VEGF165 is modulated in the presence of bFGF, IGF-1 and PlGF-1/-2. We also studied how effects of growth factor combinations can be attenuated with ranibizumab. METHODS: Effects of single growth factors or their combinations on proliferation and migration of immortalized bovine retinal endothelial cells (iBREC) were studied with or without ranibizumab or the inhibitor of VEGF receptors KRN951. RESULTS: Proliferation of iBREC was significantly stimulated by 1-100 ng/ml PlGF-1 or PlGF-2, but additive effects were not observed with various combinations of the tested growth factors. Ranibizumab neutralized VEGF's effect on proliferation but was not effective when the other growth factors were used in combination with VEGF. bFGF and IGF-1 but not PlGF-1 or PlGF-2 stimulated iBREC migration as single agents, and they further enhanced VEGF-induced migration. The effects of such growth factor combinations including VEGF on migration were efficiently blocked by targeting only VEGF with ranibizumab. Migration induced by VEGF plus bFGF and IGF-1 was also almost completely inhibited by KRN951 interfering with VEGF receptor signalling. CONCLUSIONS: Migration but not proliferation of iBREC induced by combinations of bFGF, IGF-1, PlGF-1 or PlGF-2 together with VEGF is efficiently suppressed by ranibizumab. VEGF-mediated signalling through VEGFR2 seems to control REC migration dominantly in the presence of other growth factors.

Actions of bevacizumab and ranibizumab on microvascular retinal endothelial cells: similarities and differences.

BACKGROUND: Retinal endothelial cells are crucially involved in the genesis of diabetic retinopathy which is treated with vascular endothelial growth factor (VEGF) inhibitors. Of these, ranibizumab can completely restore VEGF-induced effects on immortalised bovine retinal endothelial cells (iBREC). In most experiments supporting diabetic retinopathy therapy with bevacizumab, only non-retinal EC or retinal pigment epithelial cells have been used. Also, bevacizumab but not ranibizumab can accumulate in retinal pigment epithelial cells. OBJECTIVE: To investigate the effects of bevacizumab on VEGF-induced changes of iBREC properties and potential uptake and accumulation of both inhibitors. METHODS: Uptake of VEGF inhibitors by iBREC with or without pretreatment with VEGF(165) was visualised by immunofluorescence staining and western blot analyses. Measured transendothelial resistance (TER) of iBREC (±VEGF(165)) showed effects on permeability, indicated also by the western blot-determined tight junction protein claudin-1. The influence of bevacizumab on proliferation and migration of iBREC was studied in the presence and absence of VEGF(165). RESULTS: Bevacizumab strongly inhibited VEGF-stimulated and basal migration, but was less efficient than ranibizumab in inhibiting VEGF-induced proliferation or restoring the VEGF-induced decrease of TER and claudin-1. This ability was completely lost after storage of bevacizumab for 4 weeks at 4°C. Ranibizumab and bevacizumab were detectable in whole cell extracts after treatment for at least 1 h; bevacizumab accumulated during prolonged treatment. Ranibizumab was found in the membrane/organelle fraction, whereas bevacizumab was associated with the cytoskeleton. CONCLUSION: Both inhibitors had similar effects on retinal endothelial cells; however, some differences were recognised. Although barrier properties were not affected by internalised bevacizumab in vitro, potential adverse effects due to accumulation after repetitive intravitreal injections remain to be investigated.